Allele-specific capture of nucleic acids

ABSTRACT

A method for separating a target allele from a mixture of nucleic acids by (a) providing a mixture of nucleic acids in fluidic contact with a stabilized ternary complex that is attached to a solid support, wherein the stabilized ternary complex includes a polymerase, primed nucleic acid template, and next correct nucleotide, wherein the template has a target allele, wherein the next correct nucleotide is a cognate nucleotide for the target allele, and wherein the stabilized ternary complex is attached to the solid support via a linkage between the polymerase and the solid support or via a linkage between the next correct nucleotide and the solid support; and (b) separating the solid support from the mixture of nucleic acids, thereby separating the target allele from the mixture of nucleic acids.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.15/701,358, filed Sep. 11, 2017, which claims the benefit of U.S.Provisional Application No. 62/448,730, filed Jan. 20, 2017, which arehereby incorporated by reference in their entirety.

BACKGROUND

Small differences in nucleic acid sequences can result in significantdifferences in biological function. In the diagnostic context, singlenucleotide polymorphisms (SNPs) in the human genome underlie differencesin susceptibility to disease. A wide range of human diseases, such assickle-cell anemia, β-thalassemia, Alzheimer's and cystic fibrosisresult from SNPs.

In other contexts, single nucleotide mutations can be effective for genetherapy or synthetic biology. In gene therapy approaches a singlenucleotide change can provide a healthy version of a gene that whenintroduced to a patient's cells will treat a disease that is caused by amutant version of the gene. Synthetic biology can create industriallyuseful bio-molecules based on mutations, even at a single nucleotidesite, in the nucleic acids that encode them.

The ability to capture or select a nucleic acid having a desired SNP ormutant is useful for characterization, synthesis, and quality assessmentof nucleic acids in many contexts such as the diagnostic, therapeuticand synthetic approaches exemplified above. In many situations thedesired sequences are in low abundance and/or present in a background ofcontaminants such as other nucleic acids having different sequences.Current methods exploit the specificity of binding between complementarynucleic acid strands for such capture and selection. In a typicaltechnique, a target nucleic acid is captured using a support-boundnucleic acid that is complementary to the sequence of the target nucleicacid. Although, complementarity is theoretically capable ofdistinguishing sequences, in practical terms many samples are highlycomplex with regard to the variety of non-target sequences present andwith regard to the sheer number of non-target molecules compared totarget molecule. Such complexity makes it impractical and in some casesimprobable to selectively capture a sequence that differs fromcontaminating nucleic acids by only one or a few nucleotides.

Thus, there exists a need for methods to separate nucleic acids thatdiffer from each other by only a few or even only one nucleotide. Thepresent disclosure satisfies this need and provides related advantagesas well.

BRIEF SUMMARY

The present disclosure provides a method for separating a target allelefrom a mixture of nucleic acids. The method can include steps of (a)providing a mixture of nucleic acids in fluidic contact with astabilized ternary complex that is attached to a solid support, whereinthe stabilized ternary complex includes a polymerase, primed nucleicacid template, and next correct nucleotide, wherein the template has atarget allele, wherein the next correct nucleotide is a cognatenucleotide for the target allele, and wherein the stabilized ternarycomplex is attached to the solid support via a linkage between thepolymerase and the solid support or via a linkage between the nucleotideand the solid support; and (b) separating the solid support from themixture of nucleic acids, thereby separating the target allele from themixture of nucleic acids.

Also provided is a method for separating a first allele of a locus froma second allele at the locus. The method can include steps of (a)providing a mixture including the second allele in fluidic contact witha stabilized ternary complex that is attached to a solid support,wherein the stabilized ternary complex includes a polymerase, primerhybridized to a nucleic acid template, and next correct nucleotide,wherein the template has the first allele, wherein the next correctnucleotide is a cognate nucleotide for the first allele or the 3′ end ofthe primer has a cognate nucleotide for the first allele, and whereinthe stabilized ternary complex is attached to the solid support via alinkage between the polymerase and the solid support or via a linkagebetween the next correct nucleotide and the solid support; and (b)separating the solid support from the mixture of nucleic acids, therebyseparating the first allele from the second allele.

This disclosure further provides a method for separating a plurality oftarget alleles from a mixture of nucleic acids. The method can includesteps of (a) providing a mixture of nucleic acids in fluidic contactwith a plurality of stabilized ternary complexes that are solidsupport-attached, wherein the stabilized ternary complexes each includesa polymerase, primed nucleic acid template, and next correct nucleotide,wherein the template has a target allele, wherein the next correctnucleotide is a cognate nucleotide for the target allele, and whereineach of the stabilized ternary complexes is attached to the solidsupport via a linkage between the polymerase and the solid support orvia a linkage between the next correct nucleotide and the solid support;and (b) separating the solid support from the mixture of nucleic acids,thereby separating the target alleles from the mixture of nucleic acids.

The disclosure further provides a method for separating first alleles ata plurality of loci from second alleles at the plurality of loci,respectively. The method can include steps of (a) providing a mixture ofthe second alleles at the plurality of loci, respectively, in fluidiccontact with a plurality of stabilized ternary complexes that are solidsupport-attached, wherein the stabilized ternary complexes each includea polymerase, primed nucleic acid template, and next correct nucleotide,wherein the template includes a first allele, wherein the next correctnucleotide is a cognate nucleotide for the first allele or the 3′ end ofthe primer includes a cognate nucleotide for the first allele, andwherein each of the stabilized ternary complexes is attached to thesolid support via a linkage between the polymerase and the solid supportor via a linkage between the next correct nucleotide and the solidsupport; and (b) separating the solid support from the mixture ofnucleic acids, thereby separating the first alleles from the secondalleles at the plurality of loci.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic showing a diagrammatic representation forallele-specific ternary complex formation using a polymerase(represented as a pie shape), allele-specific primer bound to atemplate, and cognate nucleotide that binds at a position on thetemplate that is adjacent to the allele position.

FIG. 1B is a schematic showing a diagrammatic representation forallele-specific ternary complex formation using a polymerase(represented as a pie shape), allele-specific cognate nucleotide and alocus primer that binds at a region adjacent to the allele position.

FIG. 2 is a schematic showing a diagrammatic representation for a methodof separating a rare variant allele from a wild type allele thatincludes a step (1) of capturing the rare allele by formation of astabilized ternary complex with a cognate nucleotide and a biotinylatedpolymerase, followed by a step (2) of binding the biotinylatedpolymerase to a streptavidin coated bead to separate the allele-bearingternary complex from the wild type allele.

FIG. 3 is a schematic showing an exemplary workflow for separating arare variant allele from a wild type allele that includes steps of (1)annealing specific capture primer(s) (dotted lines) to a samplecontaining a mixture of nucleic acids composed of the desired target(s)as well as off-target species (solid lines); (2) adding a polymerase(pie-shape) containing a covalently attached biotin in the presence of anon-catalytic metal and cognate dNTP; (3) adding a solid capture support(partial arc of solid circle coated with streptavidin; (4) washingcaptured ternary complexes under discriminating conditions in thepresence of the non-catalytic metal and the dNTP; and (5) Eluting thetargeted nucleic acid, for example, by adding a molar excess of Mg²⁺ions or dissociating the complex.

FIG. 4 is a graph showing the results of a binding assay usingnon-labeled optical detection methods where primed template, polymeraseand nucleotide was incubated together in the presence or absence ofmagnesium.

FIG. 5 is a graph showing the effects of salt concentration on match andmismatch base discrimination effects using biolayer interferometry on aFORTEBIO® Octet instrument (Menlo Park, Calif.).

DETAILED DESCRIPTION

The present disclosure provides polymerase-based methods for selectingor capturing nucleic acids having target alleles of interest.Embodiments of the methods exploit the specificity with which apolymerase can form a stabilized ternary complex with a primed templateand a next correct nucleotide. For example, a stabilized ternary complexcan be formed between a polymerase, target allele and cognate nucleotidefor the allele. An advantage of the methods is that polymerasespecificity allows a target allele to be separated from other nucleicacids, including for example, other alleles that differ from the targetallele by a single nucleotide. For example, a ternary complex can beformed between a polymerase, a primed template encoding a target singlenucleotide polymorphism (SNP) allele and a cognate nucleotide for theSNP allele. Capture of the ternary complex will result in selectivecapture of the SNP allele, compared to a non-target SNP allele at thesame locus, because the cognate nucleotide is selective for the targetSNP when forming a ternary complex with the polymerase.

Methods and compositions set forth herein can be used to capture andoptionally enrich rare alleles (e.g. DNA- or RNA-based) containingvarious mutations within their sequences. The methods are well suited tocapture even rare variant alleles from pools of purified orsemi-purified oligonucleotides containing wild-type DNA sequences of thesame locus, as well as other unrelated sequences. FIG. 1 showsdiagrammatic representations for two different primer-nucleotidecombinations that can be used to form allele-specific ternary complexes.As shown in FIG. 1A an allele-specific primer can be used such that the3′ end of the primer is selectively matched to a target allele at aspecific locus, but mismatched to other alleles at the locus. Forexample, in the case of a single nucleotide polymorphism (SNP) locus the3′ end of the primer base-pairs with the targeted SNP allele C atposition N, but not with allele A at position N. Upon addition of apolymerase and next correct nucleotide for position N+1 (i.e. ATP in theFigure) a stabilized ternary complex can be formed selectively for thetarget allele, under conditions that do not form stabilized ternarycomplex with the mismatched, non-target allele.

Alternatively, as shown in FIG. 1B, a locus primer can be used thatbinds to multiple alleles of a particular locus, such that the 3′ end ofthe locus primer base-pairs with N−1 position. This configuration leavesthe target base of interest (at position N) available for binding to anallele specific cognate nucleotide. Again taking the example of a SNPlocus, the primer hybridizes to both alleles. Upon addition of apolymerase and the cognate nucleotide for the target SNP (i.e. thetarget SNP being C and the cognate nucleotide being GTP in the Figure),a stabilized ternary complex can be formed selectively for the targetallele, under conditions that do not form stabilized ternary complexwith the non-target, A allele.

FIG. 2 shows a diagrammatic representation for allele-specific ternarycomplex formation using a locus primer and allele-specific cognatenucleotide followed by capture of the ternary complex. The binding of abiotinylated polymerase in the presence of the correct dNTP (dCTP in theFigure) under ternary complex stabilizing conditions, will generate acapturable ternary complex with significant preference for the rareallele. The biotinylated ternary complex can be captured on magneticstreptavidin beads having affinity for the biotinylated polymerase. Thebeads and fluidic components can be separated to remove the ternarycomplex from contaminants including nucleic acids having the wild-typeallele. Optionally the beads can be washed, in the presence of the samedNTP used for capture of the rare variant allele, to further removecontaminants. Gentle elution of the nucleic acid having the rare variantallele can be achieved by contacting the ternary complex with Mg²⁻ orother ternary complex destabilizing conditions, without polymerase andwithout dNTP. The enriched allele can then be used in a variety ofdesired applications including, for example, amplification, syntheticprocedures or analytic procedures such as sequencing. Although FIG. 2 isexemplified using an allele-specific nucleotide format (e.g. as shown inFIG. 1B), it will be understood that an allele-specific primer formatcan be used as well (e.g. as shown in FIG. 1A). An exemplary workflowfor a method using allele-specific primer is shown in FIG. 3.

Generally, the methods set forth herein allow for separation of alleles.As demonstrated by the Example of FIG. 2, separation can be carried outto result in enrichment of a target allele. However, it will beunderstood that separation can be carried out to result in depletion ofa target allele. More specifically, the method shown in FIG. 2 can bemodified to capture the wild-type allele by forming a biotinylatedternary complex in the presence of dTTP instead of dCTP. Depletion ofthe wild-type allele in this way can leave behind a more enrichedpopulation of rare variant alleles. Accordingly, it will be understoodthat the term “target allele” can be used herein to refer to an allelethat is targeted for ternary complex formation in a method set forthherein independent of the desirability for using the captured alleleafterward. Thus, a target allele can be captured for purposes ofenriching the captured target allele for subsequent use or,alternatively, the target allele can be captured for purposes of beingsubsequently discarded.

It will be understood that a particular allele can be obtained using acombination of depletion and enrichment methods. For example, a rarevariant allele can be separated from a more prominent wild type alleleby subjecting a genomic sample that bears the alleles to the followingiterations. First a method set forth herein can be used to deplete thewild-type allele from the genomic sample (i.e. by forming a ternarycomplex that targets the wild type allele and then removing this ternarycomplex from the sample) and then subjecting the sample to a method setforth herein to enrich for the rare variant allele (i.e. by forming aternary complex that targets the rare variant allele and then removingthis ternary complex from the sample). In this example, depletion iscarried out prior to enrichment, but the order can be reversed ifdesired. Furthermore, the methods can be multiplexed to process aplurality of alleles in parallel.

Terms used herein will be understood to take on their ordinary meaningin the relevant art unless specified otherwise. Several terms usedherein and their meanings are set forth below.

As used herein, the term “allele,” when used in reference to a geneticlocus, refers to any of the alternative nucleotides, sequences or othergenetic features that occur at the genetic locus. Exemplary allelesinclude, but are not limited to single nucleotide polymorphisms (SNPs),insertions and/or deletions (indels), alternative mRNA splice sites orrepeats that occur at a locus.

As used herein, the term “allele-specific primer” refers to anoligonucleotide that is complementary to one allele of a locus and notto another allele of the locus. A portion of an allele-specific primercan be complementary to both alleles, so long as at least one nucleotidein the primer is a cognate for only one of the alleles. For example, anallele-specific primer can have a 3′ nucleotide that is a cognate of afirst allele at a locus, but not a cognate of a second allele at thelocus. It will be understood that an allele-specific primer can have aportion, for example, a tag or linker, that lacks complementarity toeither allele.

As used herein, the term “array” refers to a population of moleculesthat are attached to one or more solid-phase substrates such that themolecules at one feature can be distinguished from molecules at otherfeatures. An array can include different molecules that are each locatedat different addressable features on a solid-phase substrate.Alternatively, an array can include separate solid-phase substrates eachfunctioning as a feature bearing a different molecule, wherein thedifferent probe molecules can be identified according to the locationsof the solid-phase substrates on a surface to which the solid-phasesubstrates are attached or according to the locations of the solid-phasesubstrates in a liquid such as a fluid stream. The molecules of thearray can be nucleotides, nucleic acid primers, nucleic acid probes,nucleic acid templates or nucleic acid enzymes such as polymerases,ligases or exonucleases.

As used herein, the term “catalytic metal ion” refers to a metal ionthat facilitates phosphodiester bond formation between the 3′-OH of anucleic acid (e.g., a primer) and the phosphate of an incomingnucleotide by a polymerase. A “divalent catalytic metal cation” is acatalytic metal ion having a valence of two. Catalytic metal ions can bepresent at concentrations that stabilize formation of a complex betweena polymerase, nucleotide, and primed template nucleic acid, referred toas non-catalytic concentrations of a metal ion insofar as phosphodiesterbond formation does not occur. Catalytic concentrations of a metal ionrefer to the amount of a metal ion sufficient for polymerases tocatalyze the reaction between the 3′-OH group of a nucleic acid (e.g., aprimer) and the phosphate group of an incoming nucleotide.

As used herein, the term “comprising” is intended to be open-ended,including not only the recited elements, but further encompassing anyadditional elements.

As used herein, the term “each,” when used in reference to a collectionof items, is intended to identify an individual item in the collectionbut does not necessarily refer to every item in the collection.Exceptions can occur if explicit disclosure or context clearly dictatesotherwise.

As used herein, the term “exogenous,” when used in reference to a moietyof a molecule, means a chemical moiety that is not present in a naturalanalog of the molecule. For example, an exogenous label of a nucleotideis a label that is not present on a naturally occurring nucleotide.Similarly, an exogenous label that is present on a polymerase is notfound on the polymerase in its native milieu.

As used herein, the term “extension,” when used in reference to anucleic acid, refers to a process of adding at least one nucleotide tothe 3′ end of the nucleic acid. A nucleotide that is added to a nucleicacid by extension is said to be incorporated into the nucleic acid.Accordingly, the term “incorporating” can be used to refer to theprocess of joining a nucleotide to the 3′ end of a nucleic acid byformation of a phosphodiester bond.

As used herein, the term “feature” means a location in an array where aparticular molecule is present. A feature can contain only a singlemolecule or it can contain a population of several molecules of the samespecies. Alternatively, a feature can include a population of moleculesthat are different species (e.g. a population of ternary complexeshaving different template sequences). Features of an array are typicallydiscrete. The discrete features can be contiguous or they can havespaces between each other. An array useful herein can have, for example,features that are separated by less than 100 micron, 50 micron, 10micron, 5 micron, 1 micron, or 0.5 micron. Alternatively oradditionally, an array can have features that are separated by greaterthan 0.5 micron, 1 micron, 5 micron, 10 micron, 50 micron or 100 micron.The features can each have an area of less than 1 square millimeter, 500square micron, 100 square micron, 25 square micron, 1 square micron orless.

As used herein, the term “gel material” is intended to mean a semi-rigidmaterial that is permeable to liquids and gases. Typically, gel materialcan swell when liquid is taken up and can contract when liquid isremoved by drying. Exemplary gels include, but are not limited to thosehaving a colloidal structure, such as agarose; polymer mesh structure,such as gelatin; or cross-linked polymer structure, such aspolyacrylamide. Useful gels are described, for example, in US Pat. App.Pub. No. 2011/0059865 A1, and U.S. Pat. No. 9,012,022, each of which isincorporated herein by reference. The term “locus,” when used inreference to a nucleic acid, refers to the position in the nucleic acidwhere a nucleotide, nucleic acid sequence or other genetic featureoccurs.

As used herein, the term “locus-specific primer” refers to anoligonucleotide that is complementary to a first locus in a nucleic acidand not to a second locus in the nucleic acid, wherein at least twoalleles of the first locus are complementarity to the oligonucleotide.For example, the locus-specific primer can be complementary to a portionof the locus that is near or adjacent to the position of the two allelesin the nucleic acid. In the latter configuration, a locus-specificprimer can hybridize to the nucleic acid adjacent to a next templatenucleotide that is an allele. It will be understood that alocus-specific primer can have a portion, for example, a tag portion orlinker, that lacks complementarity to either locus.

As used herein, the term “next correct nucleotide” refers to thenucleotide type that will bind and/or incorporate at the 3′ end of aprimer to complement a base in a template strand to which the primer ishybridized. The base in the template strand is referred to as the “nexttemplate nucleotide” and is immediately 5′ of the base in the templatethat is hybridized to the 3′ end of the primer. The next correctnucleotide can be referred to as the “cognate” of the next templatenucleotide and vice versa. Cognate nucleotides that interact with eachother in a ternary complex or in a double stranded nucleic acid are saidto “pair” with each other. A nucleotide having a base that is notcomplementary to the next template base is referred to as an“incorrect”, “mismatch” or “non-cognate” nucleotide.

As used herein, the term “non-catalytic metal ion” refers to a metal ionthat, when in the presence of a polymerase enzyme, does not facilitatephosphodiester bond formation needed for chemical incorporation of anucleotide into a primer. A non-catalytic metal ion may interact with apolymerase, for example, via competitive binding compared to catalyticmetal ions. A “divalent non-catalytic metal ion” is a non-catalyticmetal ion having a valence of two. Examples of divalent non-catalyticmetal ions include, but are not limited to, Ca²⁺, Zn²⁺, Co²⁺, Ni²⁺, andSr²⁺. The trivalent Eu³⁺ and Tb³⁺ ions are non-catalytic metal ionshaving a valence of three.

As used herein, the term “polymerase” can be used to refer to a nucleicacid synthesizing enzyme, including but not limited to, DNA polymerase,RNA polymerase, reverse transcriptase, primase and transferase.Typically, the polymerase comprises one or more active sites at whichnucleotide binding and/or catalysis of nucleotide polymerization mayoccur. The polymerase may catalyze the polymerization of nucleotides tothe 3′ end of the first strand of the double stranded nucleic acidmolecule. For example, a polymerase catalyzes the addition of a nextcorrect nucleotide to the 3′ OH group of the first strand of the doublestranded nucleic acid molecule via a phosphodiester bond, therebycovalently incorporating the nucleotide to the first strand of thedouble stranded nucleic acid molecule. Optionally, a polymerase need notbe capable of nucleotide incorporation under one or more conditions usedin a method set forth herein. For example, a mutant polymerase may becapable of forming a ternary complex but incapable of catalyzingnucleotide incorporation.

As used herein, the term “solid support” refers to a rigid substratethat is insoluble in aqueous liquid. The substrate can be non-porous orporous. The substrate can optionally be capable of taking up a liquid(e.g. due to porosity) but will typically be sufficiently rigid that thesubstrate does not swell substantially when taking up the liquid anddoes not contract substantially when the liquid is removed by drying. Anonporous solid support is generally impermeable to liquids or gases.Exemplary solid supports include, but are not limited to, glass andmodified or functionalized glass, plastics (including acrylics,polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™,cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor,silica or silica-based materials including silicon and modified silicon,carbon, metals, inorganic glasses, optical fiber bundles, and polymers.

As used herein, the term “ternary complex” refers to an intermolecularassociation between a polymerase, a double stranded nucleic acid and anucleotide. Typically, the polymerase facilitates interaction between anext correct nucleotide and a template strand of the primed nucleicacid. A next correct nucleotide can interact with the template strandvia Watson-Crick hydrogen bonding. The term “stabilized ternary complex”means a ternary complex having promoted or prolonged existence or aternary complex for which disruption has been inhibited. Generally,stabilization of the ternary complex prevents incorporation of thenucleotide component of the ternary complex into the primed nucleic acidcomponent of the ternary complex.

The embodiments set forth below and recited in the claims can beunderstood in view of the above definitions.

The present disclosure provides a method for separating a target allelefrom a mixture of nucleic acids. The method can include steps of (a)providing a mixture of nucleic acids in fluidic contact with astabilized ternary complex that is attached to a solid support, whereinthe stabilized ternary complex includes a polymerase, primed nucleicacid template, and next correct nucleotide, wherein the template has atarget allele, wherein the next correct nucleotide is a cognatenucleotide for the target allele, and wherein the stabilized ternarycomplex is attached to the solid support via a linkage between thepolymerase and the solid support or via a linkage between the nucleotideand the solid support; and (b) separating the solid support from themixture of nucleic acids, thereby separating the target allele from themixture of nucleic acids.

Also provided is a method for separating a first allele of a locus froma second allele at the locus. The method can include steps of (a)providing a mixture including the second allele in fluidic contact witha stabilized ternary complex that is attached to a solid support,wherein the stabilized ternary complex includes a polymerase, primerhybridized to a nucleic acid template, and next correct nucleotide,wherein the template has the first allele, wherein the next correctnucleotide is a cognate nucleotide for the first allele or the 3′ end ofthe primer has a cognate nucleotide for the first allele, and whereinthe stabilized ternary complex is attached to the solid support via alinkage between the polymerase and the solid support or via a linkagebetween the next correct nucleotide and the solid support; and (b)separating the solid support from the mixture of nucleic acids, therebyseparating the first allele from the second allele.

Described herein are polymerase-based methods for capturing nucleicacids having target sequences of interest such as target alleles.Embodiments of the methods exploit the specificity with which apolymerase can form a stabilized ternary complex with the target alleleand a cognate nucleotide for the allele. The stabilized ternary complexcan include the polymerase, a primed nucleic acid template having thetarget allele, and a cognate nucleotide for the target allele. Thecognate nucleotide is not covalently attached to the primer in thestabilized ternary complex, instead being bound to the complex bynon-covalent interactions. In particular embodiments, the cognatenucleotide is non-covalently bound to the stabilized ternary complex,interacting with the other members of the complex solely vianon-covalent interactions. Useful methods and compositions for forming aternary complex are set forth in further detail below and in commonlyowned U.S. Ser. No. 14/805,381, now published as U.S. Publication No.2017/0022553 A1, and 62/375,379, which is incorporated by reference inU.S. Ser. No. 15/677,870, each of which is incorporated herein byreference.

While a ternary complex can form between a polymerase, primed templatenucleic acid and next correct nucleotide in the absence of certaincatalytic metal ions (e.g., Mg²⁺), chemical addition of the nucleotideis inhibited in the absence of the catalytic metal ions. Low ordeficient levels of catalytic metal ions, causes non-covalent (physical)sequestration of the next correct nucleotide in a stabilized ternarycomplex. Other methods disclosed herein also can be used to produce astabilized ternary complex.

Optionally, a stabilized ternary complex can be formed when the primerof the primed template nucleic acid includes a blocking group thatprecludes enzymatic incorporation of an incoming nucleotide into theprimer. The interaction can take place in the presence of stabilizers,whereby the polymerase-nucleic acid interaction is stabilized in thepresence of the next correct nucleotide (i.e., stabilizers thatstabilize the ternary complex). The primer of the primed templatenucleic acid optionally can be either an extendible primer, or a primerblocked from extension at its 3′-end (e.g., by the presence of areversible terminator moiety). The primed template nucleic acid, thepolymerase and the cognate nucleotide are capable of forming a ternarycomplex when the base of the cognate nucleotide is complementary to thenext base of the primed template nucleic acid.

As set forth above, conditions that favor or stabilize a ternary complexcan be provided by the presence of a blocking group that precludesenzymatic incorporation of an incoming nucleotide into the primer (e.g.a reversible terminator moiety on the 3′ nucleotide of the primer) orthe absence of a catalytic metal ion. Other useful conditions includethe presence of a ternary complex stabilizing agent such as anon-catalytic ion (e.g., a divalent or trivalent non-catalytic metalion) that inhibits nucleotide incorporation or polymerization. Metalions that can be used as non-catalytic metal ions for particularpolymerases include, but are not limited to, calcium, strontium,scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper,zinc, gallium, germanium, arsenic, selenium, rhodium, europium, andterbium ions. Optionally, conditions that disfavor or destabilize binarycomplexes (i.e. complexes between polymerase and primed nucleic acid butlacking cognate nucleotide) are provided by the presence of one or moremonovalent cations and/or glutamate anions. As a further option, apolymerase engineered to have reduced catalytic activity or reducedpropensity for binary complex formation can be used. Another option, isthe use of non-incorporable or non-hydrolyzable nucleotides that canform a discriminating ternary complex but cannot be incorporated intothe primer strand.

As set forth above, ternary complex stabilization conditions canaccentuate the difference in affinity of polymerase toward primedtemplate nucleic acids in the presence of different nucleotides, forexample, by destabilizing binary complexes. Optionally, the conditionscause differential affinity of the polymerase for the primed templatenucleic acid in the presence of different nucleotides. By way ofexample, the conditions include, but are not limited to, high salt andglutamate ions. For example, the salt may dissolve in aqueous solutionto yield a monovalent cation, such as a monovalent metal cation (e.g.,sodium ion or potassium ion). Optionally, the salt that provides themonovalent cations (e.g., monovalent metal cations) further providesglutamate anions. Optionally, the source of glutamate ions can bepotassium glutamate. In some instances, the concentrations of potassiumglutamate that can be used to alter polymerase affinity of the primedtemplate nucleic acid extend from 10 mM to 1.6 M of potassium glutamate,or any amount in between 10 mM and 1.6 M. As indicated above, high saltrefers to a concentration of salt from 50 to 1,500 mM salt.

It will be understood that options set forth herein for stabilizing aternary complex need not be mutually exclusive and instead can be usedin various combinations. For example, a ternary complex can bestabilized by one or a combination of means including, but not limitedto, crosslinking of the polymerase domains, crosslinking of thepolymerase to the nucleic acid, polymerase mutations that stabilize theternary complex, allosteric inhibition by small molecules, uncompetitiveinhibitors, competitive inhibitors, non-competitive inhibitors, andother means set forth herein.

A ternary complex that is made or used in accordance with the presentdisclosure may optionally include one or more exogenous label. The labelcan be present on the polymerase, template nucleic acid, primer and/orcognate nucleotide. Exogenous labels can be useful for detecting aternary complex or an individual component thereof, during one or moreof the manipulations set forth herein. Exemplary labels, methods forattaching labels and methods for using labeled components are set forthin commonly owned U.S. Ser. No. 14/805,381, now published as U.S.Publication No. 2017/0022553, and 62/375,379, which is incorporated byreference in U.S. Ser. No. 15/677,870, each of which is incorporatedherein by reference.

In alternative embodiments, a ternary complex can lack exogenous labels.For example, a ternary complex and components used in the formation ofthe ternary complex (e.g. polymerase, template nucleic acid, primerand/or cognate nucleotide) can lack one, several or all of the exogenouslabels described in the above-incorporated references.

A ternary complex can be formed with any of a variety of nucleic acidtemplate sequences in a method set forth herein. The methods areparticularly useful for selectively capturing one allele at a geneticlocus to separate it from one or more other alleles at the locus. Thus,a mixture of nucleic acid templates that is used in a method set forthherein can include first and second alleles at a particular locus, oneof which is selectively captured. The mixture can include a variety ofother nucleic acids, for example, some or all of the sequence content ofa genome or exome from one or more organism.

Methods set forth herein can be particularly useful for selectivelycapturing a minor allele. The minor allele can be one of a pairoccurring at bi-allelic locus, one of three alleles at a tri-alleliclocus or one of four alleles at a quad-allelic locus. The minor allelefrequency of an allele captured herein can be at most 40%, 25%, 10%, 5%,0.5% or less. The methods can also be used to capture alleles havinghigher frequency including, for example, major alleles. Exemplaryalleles that can be captured include, without limitation, singlenucleotide polymorphisms (SNPs), insertion-deletion (indel)polymorphisms and alternative splicing polymorphisms.

Although the methods of the present disclosure are particularly wellsuited to selectively capturing an allele at a multi-allelic locus,other sequences can also be captured. Thus, the methods and compositionsexemplified for alleles can be extended to other sequences and othertemplates. For example, the methods can be used to capture anon-polymorphic sequence. In such cases the next correct nucleotide andprimer need not correlate to a particular allele in the template. Inother embodiments, the methods can be used to selectively capture amutant sequence compared to its wild-type sequence or vice versa. Thiscan be useful for example, when manipulating or evaluating reagents foror products of protein engineering or synthetic biology.

Nucleic acid templates that are used in a method or composition hereincan be DNA such as genomic DNA, synthetic DNA, amplified DNA,complementary DNA (cDNA) or the like. RNA can also be used such as mRNA,ribosomal RNA, tRNA or the like. Nucleic acid analogs can also be usedas templates herein. Thus, a mixture of nucleic acids used herein can bederived from a biological source, synthetic source or amplificationproduct. Primers used herein can be DNA, RNA or analogs thereof.

Exemplary organisms from which nucleic acids can be derived include, forexample, those from a mammal such as a rodent, mouse, rat, rabbit,guinea pig, ungulate, horse, sheep, pig, goat, cow, cat, dog, primate,human or non-human primate; a plant such as Arabidopsis thaliana, corn,sorghum, oat, wheat, rice, canola, or soybean; an algae such asChlamydomonas reinhardtii; a nematode such as Caenorhabditis elegans; aninsect such as Drosophila melanogaster, mosquito, fruit fly, honey beeor spider; a fish such as zebrafish; a reptile; an amphibian such as afrog or Xenopus laevis; a dictyostelium discoideum; a fungi such aspneumocystis carinii, Takifugu rubripes, yeast, Saccharamoycescerevisiae or Schizosaccharomyces pombe; or a plasmodium falciparum.Nucleic acids can also be derived from a prokaryote such as a bacterium,Escherichia coli, staphylococci or mycoplasma pneumoniae; an archae; avirus such as Hepatitis C virus or human immunodeficiency virus; or aviroid. Nucleic acids can be derived from a homogeneous culture orpopulation of the above organisms or alternatively from a collection ofseveral different organisms, for example, in a community or ecosystem.Nucleic acids can be isolated using methods known in the art including,for example, those described in Sambrook et al., Molecular Cloning: ALaboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, New York(2001) or in Ausubel et al., Current Protocols in Molecular Biology,John Wiley and Sons, Baltimore, Md. (1998), each of which isincorporated herein by reference.

Particular embodiments of the methods set forth herein can use a nativenucleotide, nucleotide analog or modified nucleotide. Such nucleotidescan be used, for example, for forming a stabilized ternary complex.Optionally, a nucleotide analog comprises a nitrogenous base,five-carbon sugar, and phosphate group; wherein any moiety of thenucleotide may be modified, removed and/or replaced. Nucleotide analogsmay be non-incorporable nucleotides. Such nucleotides incapable ofincorporation include, for example, monophosphate and diphosphatenucleotides. In another example, the nucleotide may containmodification(s) to the triphosphate group that make the nucleotidenon-incorporable. Examples of non-incorporable nucleotides may be foundin U.S. Pat. No. 7,482,120, which is incorporated by reference herein.Non-incorporable nucleotides may be subsequently modified to becomeincorporable. Nucleotide analogs include, but are not limited to,alpha-phosphate modified nucleotides, alpha-beta nucleotide analogs,beta-phosphate modified nucleotides, beta-gamma nucleotide analogs,gamma-phosphate modified nucleotides, caged nucleotides, or ddNTPs.Examples of nucleotide analogs are described in U.S. Pat. No. 8,071,755,which is incorporated by reference herein.

Nucleotide analogs can include terminators that reversibly preventnucleotide incorporation at the 3′-end of the primer after the analoghas been incorporated. For example, U.S. Pat. No. 7,544,794 and U.S.Pat. No. 8,034,923 (the disclosures of these patents are incorporatedherein by reference) describe reversible terminators in which the 3′-OHgroup is replaced by a 3′-ONH₂ moiety. Another type of reversibleterminator is linked to the nitrogenous base of a nucleotide as setforth, for example, in U.S. Pat. No. 8,808,989 (the disclosure of whichis incorporated herein by reference). Other reversible terminators thatsimilarly can be used in connection with the methods described hereininclude those described in U.S. Pat. Nos. 7,956,171, 8,071,755, and9,399,798 (the disclosures of these U.S. patents are incorporated hereinby reference). In certain embodiments, a reversible blocking moiety canbe removed from a primer, allowing for nucleotide incorporation.Compositions and methods for deblocking are set forth in the abovereferences.

Alternatively, nucleotide analogs irreversibly prevent nucleotideincorporation at the 3′-end of the primer to which they have beenincorporated. Irreversible nucleotide analogs include 2′,3′-dideoxynucleotides, ddNTPs (ddGTP, ddATP, ddTTP, ddCTP).Dideoxynucleotides lack the 3′-OH group of dNTPs that is essential forpolymerase-mediated primer extension.

Optionally, a nucleotide (e.g. a native nucleotide or nucleotide analog)is present in a mixture during formation of a stabilized ternarycomplex. For example, at least 1, 2, 3, 4 or more nucleotide types canbe present. Alternatively or additionally, at most 4, 3, 2, or 1nucleotide types can be present. Similarly, one or more nucleotide typesthat are present can be complementary to at least 1, 2, 3 or 4nucleotide types in a template nucleic acid. Alternatively oradditionally, one or more nucleotide types that are present can becomplementary to at most 4, 3, 2, or 1 nucleotide types in a templatenucleic acid.

Optionally, a nucleotide analog is fused to a polymerase. Optionally, aplurality of nucleotide analogs comprises fusions to a plurality ofpolymerases, wherein each nucleotide analog comprises a differentpolymerase.

Any nucleotide modification that stabilizes a polymerase in a ternarycomplex may be used in the methods disclosed herein. The nucleotide maybe bound permanently or transiently. Optionally, a nucleotide that ispresent in a stabilized ternary complex is not the means by which theternary complex is stabilized. Accordingly, any of a variety of otherternary complex stabilization methods may be combined in a reactionutilizing a nucleotide analog.

In particular embodiments, the primer strand of a primed templatenucleic acid molecule undergoing one or more steps of a method set forthherein is chemically unchanged by the polymerase. This is to say thatthe primer is neither extended by formation of a new phosphodiesterbond, nor shortened by nucleolytic degradation during the examinationstep to identify the next correct nucleotide.

Polymerases that may be used to carry out a method of the presentdisclosure include naturally occurring polymerases and modifiedvariations thereof, including, but not limited to, mutants,recombinants, fusions, genetic modifications, chemical modifications,synthetics, and analogs. Naturally occurring polymerases and modifiedvariations thereof are not limited to polymerases that retain theability to catalyze a polymerization reaction. Optionally, the naturallyoccurring and/or modified variations thereof retain the ability tocatalyze a polymerization reaction. Optionally, the naturally-occurringand/or modified variations have special properties, for example,enhanced binding affinity to nucleic acids, reduced binding affinity tonucleic acids, enhanced binding affinity to nucleotides, reduced bindingaffinity to nucleotides, enhanced specificity for next correctnucleotides, reduced specificity for next correct nucleotides, enhancedcatalysis rates, reduced catalysis rates, catalytic inactivity etc.Mutant polymerases include polymerases wherein one or more amino acidsare replaced with other amino acids, and insertions or deletions of oneor more amino acids.

Modified polymerases include polymerases that contain an exogenousaffinity moiety (e.g., an exogenous ligand or receptor), which can beused to capture or manipulate the polymerase. Optionally, the affinitymoiety can be attached after the polymerase has been at least partiallypurified using protein isolation techniques. For example, the exogenousaffinity moiety can be chemically linked to the polymerase using a freesulfhydryl or a free amine moiety of the polymerase. This can involvechemical linkage to the polymerase through the side chain of a cysteineresidue, or through the free amino group of the N-terminus. An exogenousaffinity moiety can also be attached to a polymerase via protein fusion.Exemplary affinity moiety that can be attached via protein fusioninclude, for example, poly histidine, antibody fragments, epitopes forparticular antibodies, streptavidin and affinity tags used forpurification of recombinant proteins (e.g. commercially availableaffinity tags from ThermoFisher, Waltham, Mass. or Promega, MadisonWis.).

Useful DNA polymerases include, but are not limited to, bacterial DNApolymerases, eukaryotic DNA polymerases, archaeal DNA polymerases, viralDNA polymerases and phage DNA polymerases. Bacterial DNA polymerasesinclude E. coli DNA polymerases I, II and III, IV and V, the Klenowfragment of E. coli DNA polymerase, Clostridium stercorarium (Cst) DNApolymerase, Clostridium thermocellum (Cth) DNA polymerase and Sulfolobussolfataricus (Sso) DNA polymerase. Eukaryotic DNA polymerases includeDNA polymerases α, β, γ, δ, €, η, ζ, λ, σ, μ, and k, as well as the Rev1polymerase (terminal deoxycytidyl transferase) and terminaldeoxynucleotidyl transferase (TdT). Viral DNA polymerases include T4 DNApolymerase, phi-29 DNA polymerase, GA-1, phi-29-like DNA polymerases,PZA DNA polymerase, phi-15 DNA polymerase, Cpl DNA polymerase, Cp1 DNApolymerase, T7 DNA polymerase, and T4 polymerase. Other useful DNApolymerases include thermostable and/or thermophilic DNA polymerasessuch as Thermus aquaticus (Taq) DNA polymerase, Thermus filiformis (Tfi)DNA polymerase, Thermococcus zilligi (Tzi) DNA polymerase, Thermusthermophilus (Tth) DNA polymerase, Thermus flavusu (Tfl) DNA polymerase,Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcus furiosus (Pfu) DNApolymerase and Turbo Pfu DNA polymerase, Thermococcus litoralis (Tli)DNA polymerase, Pyrococcus sp. GB-D polymerase, Thermotoga maritima(Tma) DNA polymerase, Bacillus stearothermophilus (Bst) DNA polymerase,Pyrococcus Kodakaraensis (KOD) DNA polymerase, Pfx DNA polymerase,Thermococcus sp. JDF-3 (JDF-3) DNA polymerase, Thermococcus gorgonarius(Tgo) DNA polymerase, Thermococcus acidophilium DNA polymerase;Sulfolobus acidocaldarius DNA polymerase; Thermococcus sp. go N-7 DNApolymerase; Pyrodictium occultum DNA polymerase; Methanococcus voltaeDNA polymerase; Methanococcus thermoautotrophicum DNA polymerase;Methanococcus jannaschii DNA polymerase; Desulfurococcus strain TOK DNApolymerase (D. Tok Pol); Pyrococcus abyssi DNA polymerase; Pyrococcushorikoshii DNA polymerase; Pyrococcus islandicum DNA polymerase;Thermococcus fumicolans DNA polymerase; Aeropyrum pernix DNA polymerase;and the heterodimeric DNA polymerase DP1/DP2. Engineered and modifiedpolymerases also are useful in connection with the disclosed techniques.For example, modified versions of the extremely thermophilic marinearchaea Thermococcus species 9° N (e.g., Therminator DNA polymerase fromNew England BioLabs Inc.; Ipswich, Mass.) can be used. Still otheruseful DNA polymerases, including the 3PDX polymerase are disclosed inU.S. Pat. No. 8,703,461, the disclosure of which is incorporated hereinby reference.

Useful RNA polymerases include, but are not limited to, viral RNApolymerases such as T7 RNA polymerase, T3 polymerase, SP6 polymerase,and K11 polymerase; Eukaryotic RNA polymerases such as RNA polymerase I,RNA polymerase II, RNA polymerase III, RNA polymerase IV, and RNApolymerase V; and Archaea RNA polymerase.

Useful reverse transcriptases include, but are not limited to, HIV-1reverse transcriptase from human immunodeficiency virus type 1 (PDB1HMV), HIV-2 reverse transcriptase from human immunodeficiency virustype 2, M-MLV reverse transcriptase from the Moloney murine leukemiavirus, AMV reverse transcriptase from the avian myeloblastosis virus,and Telomerase reverse transcriptase that maintains the telomeres ofeukaryotic chromosomes.

A polymerase having an intrinsic 3′-5′ proofreading exonuclease activitycan be useful for some embodiments. Polymerases that substantially lack3′-5′ proofreading exonuclease activity are also useful in someembodiments, for example, in most sequencing embodiments. Absence ofexonuclease activity can be a wild type characteristic or acharacteristic imparted by a variant or engineered polymerase structure.For example, exo minus Klenow fragment is a mutated version of Klenowfragment that lacks 3′-5′ proofreading exonuclease activity. Klenowfragment and its exo minus variant can be useful in a method orcomposition set forth herein.

A stabilized ternary complex can be attached to a solid support. Thesolid support can be made from any of a variety of materials set forthherein, for example, above in the definitions or below. Suitablematerials may include glass, polymeric materials, silicon, quartz (fusedsilica), borofloat glass, silica, silica-based materials, carbon,metals, an optical fiber or bundle of optical fibers, sapphire, orplastic materials. The particular material can be selected based onproperties desired for a particular use. For example, materials that aretransparent to a desired wavelength of radiation are useful foranalytical techniques that will utilize radiation of that wavelength.Conversely, it may be desirable to select a material that does not passradiation of a certain wavelength (e.g. being opaque, absorptive orreflective). Other properties of a material that can be exploited areinertness or reactivity to certain reagents used in a downstreamprocess, such as those set forth herein; or ease of manipulation or lowcost of manufacture.

A particularly useful solid support is a particle such as a bead ormicrosphere as exemplified below. Populations of beads can be used forattachment of populations of stabilized ternary complexes. In someembodiments it may be useful to use a configuration whereby each beadhas a single type of stabilized ternary complex (e.g. one allele typeper bead). Alternatively, different stabilized ternary complexes neednot be separated on a bead-by-bead basis. As such a bead can bearmultiple different types of stabilized ternary complexes (e.g. multipletypes of alleles per bead). The composition of a bead can vary,depending for example, on the format, chemistry and/or method ofattachment to be used. Exemplary bead compositions include solidsupports, and chemical functionalities imparted thereto, used in proteinand nucleic acid capture methods. Such compositions include, forexample, plastics, ceramics, glass, polystyrene, melamine,methylstyrene, acrylic polymers, paramagnetic materials, thoria sol,carbon graphite, titanium dioxide, latex or cross-linked dextrans suchas Sepharose™, cellulose, nylon, cross-linked micelles and Teflon™, aswell as other materials set forth in “Microsphere Detection Guide” fromBangs Laboratories, Fishers Ind., which is incorporated herein byreference.

The geometry of a particle, bead or microsphere also can correspond to awide variety of different forms and shapes. For example, they can besymmetrically shaped (e.g. spherical or cylindrical) or irregularlyshaped (e.g. controlled pore glass). In addition, beads can be, forexample, porous, thus increasing the surface area available for captureof ternary complexes or components thereof. Exemplary sizes for beadsused herein can range from nanometers to millimeters or from about 10nm-1 mm.

In particular embodiments, beads can be arrayed or otherwise spatiallydistinguished. Exemplary bead-based arrays that can be used include,without limitation, those in which beads are associated with a solidsupport such as those described in U.S. Pat. No. 6,355,431 B1, U.S. Pub.No. 2002/0102578 or PCT Pub. No. WO 00/63437, each of which isincorporated herein by reference. Beads can be located at discretelocations, such as wells, on a solid-phase support, whereby eachlocation accommodates a single bead. Alternatively, discrete locationswhere beads reside can each include a plurality of beads as described,for example, in U.S. Pub. Nos. 2004/0263923, 2004/0233485, 2004/0132205,or 2004/0125424, each of which is incorporated herein by reference.

Another useful solid support is an array of features. Arrays areparticularly useful for multiplex applications wherein a plurality ofdifferent ternary complexes are made and used. Compositions andtechniques for making and using arrays are set forth in further detailbelow. Features on an array can be used for attachment of populations ofstabilized ternary complexes. In some embodiments, it may be useful touse a configuration whereby each feature has a single type of stabilizedternary complex (e.g. one allele type per feature). Alternatively,different stabilized ternary complexes need not be separated on afeature-by-feature basis. As such, a feature can bear multiple differenttypes of stabilized ternary complexes (e.g. multiple types of allelesper feature).

A stabilized ternary complex or component that is used to make such acomplex can be attached to a solid support using any of a variety ofmethods well known in the art. Such methods include for example,attachment by direct chemical synthesis onto the solid support, chemicalattachment, photochemical attachment, thermal attachment, enzymaticattachment and/or absorption. These and other methods are well known inthe art and applicable for attachment of proteins, nucleotides ornucleic acids in any of a variety of formats and configurations.Attachment to a solid support can occur via a covalent linkage or vianon-covalent interactions. Exemplary non-covalent interactions are thosebetween a ligand-receptor pair such as streptavidin (or analogs thereof)and biotin (or analogs thereof) or between an antibody (or functionalfragment thereof such as a Fab or ScFv) and epitope. Other usefulreceptor-ligand pairs include lectin and carbohydrate, and complementaryfirst and second strands of a nucleic acid.

A polymerase, nucleotide, primer or template that participates information of a ternary complex can be attached to a solid support eitherbefore or after formation of a stabilized ternary complex. An exemplaryembodiment wherein a ternary complex is formed in solution andsubsequently attached to a solid support is diagramed in FIG. 2. Asexemplified in the Figure, a polymerase can include a ligand moiety(e.g. biotin) that is bound to a solid-phase receptor (e.g.streptavidin) after a stabilized ternary complex is formed.Alternatively, it is possible to bind a ligand moiety of a polymerase toa solid phase receptor (e.g. by binding a biotinylated polymerase tostreptavidin beads) prior to formation of the stabilized ternarycomplex. Thus, a stabilized ternary complex can be formed on a solidsupport.

Although variability in the order of solid-phase attachment and ternarycomplex formation is exemplified above for attachment via polymerase, itwill be understood that similar variability in the order of steps canoccur when other components of the ternary complex have the attachmentmoiety. Similar variability in the order of steps can occur usinglinkages other than receptor-ligand interactions. For example, gentlechemistry conditions can be used that allow a chemical attachment moietyto bond covalently with a solid support before or after formation of aternary complex. Exemplary chemistry conditions and linkages includethose used routinely for modifying active proteins and enzymes such asthose commercially available from ThermoFisher (Waltham, Mass.), SigmaAldrich (St. Louis, Mo.) or Promega (Madison Wis.).

Other chemistry conditions and linkages that are useful are those knownas “click chemistry” (e.g. U.S. Pat. No. 6,737,236 and U.S. Pat. No.7,427,678, each incorporated herein by reference in its entirety). Alsouseful are azide alkyne Huisgen cycloaddition reactions, which use acopper catalyst (e.g. U.S. Pat. Nos. 7,375,234 and 7,763,736, eachincorporated herein by reference in its entirety). Copper-free Huisgenreactions (“metal-free click”) using strained alkynes can be employed.Other useful linkage chemistries include, but are not limited totriazine-hydrazine moieties which can link to aldehyde moieties, forexample as described in U.S. Pat. No. 7,259,258, which is incorporatedby reference; triazine chloride moieties which can link to aminemoieties; carboxylic acid moieties which can link to amine moietiesusing a coupling reagent, such as EDC, thiol moieties which can link tothiol moieties; alkene moieties which can link to dialkene moieties thatare coupled through Diels-Alder reactions; and acetyl bromide moietieswhich can link thiophosphate moieties, such as those described in PCTPub. No. WO 2005/065814, which is incorporated by reference. Glass-likesurfaces can also be modified with various glass-reactive molecules,such as functionalized silanes, some of which are commercially availablethrough Gelest, Inc.

Accordingly, a method for separating a target allele from a mixture ofnucleic acids can include steps of (a) (i) providing a mixture ofnucleic acids including a primed template nucleic acid and contactingthe mixture with a polymerase and a next correct nucleotide, wherein thetemplate has a target allele, wherein the next correct nucleotide is acognate nucleotide for the target allele, and (ii) forming a stabilizedternary complex, wherein the stabilized ternary complex includes thepolymerase, primed nucleic acid template, and next correct nucleotide,and wherein the stabilized ternary complex is attached to the solidsupport via a linkage between the polymerase and the solid support orwherein the stabilized ternary complex is attached to the solid supportvia a linkage between the next correct nucleotide and the solid support;and (b) separating the solid support from the mixture of nucleic acids,thereby separating the target allele from the mixture of nucleic acids.

Optionally, the polymerase or next correct nucleotide is attached to thesolid support prior to forming the stabilized ternary complex on thesolid support in step (a)(ii). As such, stabilized ternary complex isformed on the solid support. In an alternative option, the polymerase ornext correct nucleotide is attached to the solid support after thestabilized ternary complex is formed during or after step (a)(ii). Thus,the ternary complex is formed in solution prior to attaching the ternarycomplex to the solid support. The latter option is exemplified in FIG.2.

A method of the present disclosure can include a step of separating asolid support, to which a stabilized ternary complex is attached, from amixture of nucleic acids. Thus, an allele that was in the mixture, andsubsequently attached to the ternary complex, can be separated from themixture. Separation can be carried out using a method appropriate forthe solid support. For example, when a magnetic support is used, forexample as shown in FIG. 2, a magnet can be used to attract the supportfor purposes of separation (e.g. the magnet can be used to remove thesupport from a vessel containing the mixture or the magnet can be usedto retain the solid support in a vessel from which the mixture isremoved). Similarly, beads or other particles can be separated from amixture based on other attractive forces such as the force of gravity(e.g. settling or centrifugation) on materials that are denser than themixture, electrical attraction of charged materials, optical attractionof dielectric particles, or affinity attraction of the particles toother surfaces (e.g. via receptor-ligand interactions).

Separation of a solid support-bound ternary complex from a fluidicmixture can also be achieved by fluidic flow of the fluid away from thesurface of the solid support. For example, a stabilized ternary complexcan be attached to a surface of a vessel, such as a flow cell or arraysurface, and the fluidic mixture can be removed from contact with thesurface via gravity (e.g. pouring), pump action (e.g. positive pressureor negative pressure), capillary action, electrophoresis, digitalfluidics whereby droplets are moved by electrowetting or other forces(see, for example, US Pub. Nos. 2007/0242105; 2011/0303542 or2008/0283414), or the like.

In particular embodiments, a stabilized ternary complex is attached to aflow cell surface or to a solid support in a flow cell. A flow cellallows convenient fluidic manipulation by passing solutions into and outof a fluidic chamber that contacts the support-bound, ternary complex.Exemplary flow cells that can be used are described, for example, inU.S. Pub. Nos. 2010/0111768 A1, WO 05/065814 and US Pub. No.2012/0270305, each of which is incorporated herein by reference.

Optionally, the provided methods further include a wash step. The washstep can occur before or after any other step in the method. Forexample, a method set forth herein can optionally include a step ofwashing a solid support that is attached to a stabilized ternarycomplex. The wash can provide the advantage of removing contaminantssuch as components of a mixture from which one or more components of thestabilized ternary complex were derived. In particular embodiments, thewash step occurs under conditions that stabilize the ternary complex.For example, one or more of the stabilizing conditions or stabilizingagents set froth elsewhere herein can be employed during a wash step.Optionally, the wash solution includes nucleotide(s) of the same type asthe next correct nucleotide(s) used during formation of the stabilizedternary complex. Including the next correct nucleotide(s) at asufficient concentration can provide the advantage of stabilizingpreviously formed ternary complexes from unwanted disassociation. Thisin turn prevents unwanted loss of target allele due to washing awaypreviously formed ternary complexes. Optionally, the ternary complex hasa half-life and the wash step is performed for a duration shorter thanthe half-life of the ternary complex.

A method of the present disclosure can further include a step ofdissociating a target allele from a solid support. For example,dissociation can occur after a solid support-bound, stabilized ternarycomplex has been removed from a mixture where the complex was formed.Optionally, dissociation can be carried without covalently adding thenext correct nucleotide to the 3′ end of the primer. This can beachieved by maintaining ternary complex stabilization until the primeris no longer present in the ternary complex. An advantage of releasingunmodified primer is that the primer can be re-used for capturing thesame type of allele. For example, the primer can be used in an iterativemethod to recapture the same allele as set forth in further detailbelow. The primer can also be used with a new mixture of nucleic acidsto capture a new template nucleic acid having the same type of allele.

Exemplary dissociation techniques include, but are not limited to,denaturation of the polymerase, competitive binding of a differentnucleic acid to the polymerase to cause release of the target allele,incubation of the ternary complex in a solution that is devoid of nextcorrect nucleotide, in a solution that is devoid of primed template, orin a solution having a concentration of next correct nucleotide orprimed template that is substantially below the dissociation constant(K_(d)) of the polymerase for the next correct nucleotide or primedtemplate, respectively. In some embodiments, the ternary complex can beincubated with a nucleotide that is different from the next correctnucleotide (e.g. a cognate nucleotide for a different allele than thetarget allele). This dissociation method provides an advantage of beingrelatively gentle and specific such that dissociation of the desiredallele is selected over other alleles that may be present ascontaminants.

Alternatively, a step of dissociating a target allele from a solidsupport can be carried out by extending the primer to incorporate a nextcorrect nucleotide. The nucleotide that is incorporated can be anucleotide molecule that was present in the stabilized ternary complexwhen it was formed in a mixture and/or when the complex was separatedfrom the mixture. Alternatively, a different nucleotide molecule canenter the ternary complex and then be incorporated into the primer.Thus, the incorporation step can involve replacing a nucleotide from aprior step and incorporating another nucleotide into the 3′-end of theprimer. The incorporation step can involve releasing a nucleotide fromwithin a ternary complex and incorporating a nucleotide of a differentkind into the 3′-end of the primer.

Optionally, the nucleotide that is incorporated can have an exogenouslabel. An advantage of using a label is the ability to confirm theidentity of the next correct nucleotide by detecting the label on theprimer. Alternatively, the nucleotide that is incorporated will lack anexogenous label that is detected.

Accordingly, the methods described herein optionally include anincorporation step. The incorporation step involves covalentlyincorporating one or more nucleotides at the 3′-end of a primerhybridized to a template nucleic acid. In some embodiments, only asingle nucleotide is incorporated at the 3′-end of the primer. Forexample, the 3′ position of the nucleotide can be modified to include a3′ terminator moiety. The 3′ terminator moiety may be a reversibleterminator or may be an irreversible terminator. Optionally, thereversible terminator nucleotide includes a 3′-ONH₂ moiety attached atthe 3′ position of the sugar moiety. Further examples of usefulreversible terminator moieties are described, for example, in Bentley etal., Nature 456:53-59 (2008), WO 04/018497; U.S. Pat. No. 7,057,026; WO91/06678; WO 07/123744; U.S. Pat. No. 7,329,492; U.S. Pat. No.7,211,414; U.S. Pat. No. 7,315,019; U.S. Pat. No. 7,405,281, and US2008/0108082, each of which is incorporated herein by reference.Optionally, multiple nucleotides are incorporated at the 3′-end of theprimer. For example, the nucleotide that is incorporated can include a3′-hydroxyl group that is capable of being further extended afterincorporation. In some embodiments, the incorporation step is part of asequencing technique, amplification technique, or other techniquecarried out using ternary complex that has been captured using a methodset forth herein.

Incorporated nucleotides alternatively can be unlabeled nucleotides, ordetectably labeled nucleotide analogs. Whether labeled or not, thenucleotides can be terminator nucleotides that are permanently orreversibly prevented from being extended once incorporated into aprimer. The polymerase can dissociate from primed template afternucleotide incorporation. Exemplary reagents and conditions forincorporating nucleotides into the primed template of a ternary complexare set forth in commonly owned U.S. Pat. App. Ser. No. 14/805,381, nowpublished as US Pub. No. 2017/0022553, and 62/375,379, which isincorporated by reference in U.S. Ser. No. 15/677,870, each of which isincorporated herein by reference.

A target allele or other target sequence can be captured using aniterative method whereby steps of a method set forth herein arerepeated. The methods set forth herein are well suited to iterationbecause the nucleic acid primer need not be consumed or modifiedfollowing use to capture a target sequence. This contrasts with othermethods of nucleic acid capture where a primer is extended toincorporate an affinity labeled nucleotide. Once the primer has beenmodified in this way it has been spent and must be replaced orchemically reverted for use in a repetition of the primer extensionstep.

An advantage of the iterative approach provided by the currentdisclosure is that each iteration can further purify the target sequenceallowing the nucleic acid to be isolated from other biological materialsincluding other nucleic acids having similar sequences (e.g. otheralleles at the same locus as the target allele). The reagents can bere-used thereby providing a cost- and time-effective alternative toother methods that consume primers and other reagents.

Accordingly, the present disclosure provides a method for separating atarget allele from a mixture of nucleic acids. The method can includesteps of (a) providing a mixture of nucleic acids in fluidic contactwith a stabilized ternary complex that is attached to a solid support,wherein the stabilized ternary complex includes a polymerase, primednucleic acid template, and next correct nucleotide, wherein the templatehas a target allele, wherein the next correct nucleotide is a cognatenucleotide for the target allele, and wherein the stabilized ternarycomplex is attached to the solid support via a linkage between thepolymerase and the solid support or via a linkage between the nucleotideand the solid support; (b) separating the solid support from the mixtureof nucleic acids, thereby separating the target allele from the mixtureof nucleic acids; (c) dissociating the template having the target allelefrom the separated solid support; (d) forming a solution including asecond stabilized ternary complex that includes the formerly dissociatedtemplate, a polymerase, and a next correct nucleotide that is a cognatenucleotide for the target allele, wherein the polymerase or the nextcorrect nucleotide is attached to a solid support; and (e) separatingthe solid support of step (d) from the solution, thereby separating theformerly dissociated template having the target allele from thesolution. The method can further include an optional step: (f)dissociating the formerly dissociated template from the solid supportseparated in step (e).

Also provided is a method for separating a first allele of a locus froma second allele at the locus. The method can include steps of (a)providing a mixture including the second allele in fluidic contact witha stabilized ternary complex that is attached to a solid support,wherein the stabilized ternary complex includes a polymerase, primerhybridized to a nucleic acid template, and next correct nucleotide,wherein the template has the first allele, wherein the next correctnucleotide is a cognate nucleotide for the first allele or the 3′ end ofthe primer has a cognate nucleotide for the first allele, and whereinthe stabilized ternary complex is attached to the solid support via alinkage between the polymerase and the solid support or via a linkagebetween the next correct nucleotide and the solid support; (b)separating the solid support from the mixture of nucleic acids, therebyseparating the first allele from the second allele; (c) dissociating thetemplate having the first allele from the separated solid support; (d)forming a solution including a second stabilized ternary complex thatincludes the formerly dissociated template, a polymerase, and a nextcorrect nucleotide, wherein the polymerase or the next correctnucleotide is attached to a solid support; and (e) separating the solidsupport of step (d) from the solution, thereby separating the formerlydissociated template having the first allele from the solution. Themethod can further include an optional step: (f) dissociating theformerly dissociated template from the solid support separated in step(e).

In particular embodiments of the above iterative methods, the same solidsupport is used for step (d) as was used in step (a). This can providecost savings compared to using a new solid support in step (d).Nevertheless, in some embodiments different solid supports are used insteps (d) and (a). The solid supports used in each step can be of thesame type or different type. Any of a variety of solid supports setforth herein or otherwise known in the art can be used.

Similarly, the same polymerase can optionally be used for step (d) aswas used in step (a). Again, this can provide cost savings compared tousing a new polymerase in step (d). Nevertheless, in some embodimentsdifferent polymerases are used in steps (d) and (a). The polymerasesused in each step can be of the same type or different type. Any of avariety of polymerases set forth herein or otherwise known in the artcan be used.

Typically, the same primer is used for step (a) and step (d). However,in some embodiments, a new primer can be used in step (d). The newprimer will typically be of the same type as the previously used primer(i.e. the two primers can have the same sequence and be of the samelength). However, if desired primers of different length or sequence canbe used.

A method of the present disclosure can be carried out in a multiplexformat whereby multiple different types of nucleic acids are processedin parallel during one or more steps set forth herein. Although it isalso possible to serially process different types of nucleic acids usingone or more steps of the methods set forth herein, parallel processingcan provide cost savings, time savings and uniformity of conditions.

Accordingly, this disclosure provides a method for separating aplurality of target alleles from a mixture of nucleic acids. The methodcan include steps of (a) providing a mixture of nucleic acids in fluidiccontact with a plurality of stabilized ternary complexes that are solidsupport-attached, wherein the stabilized ternary complexes each includesa polymerase, primed nucleic acid template, and next correct nucleotide,wherein the template has a target allele, wherein the next correctnucleotide is a cognate nucleotide for the target allele, and whereineach of the stabilized ternary complexes is attached to the solidsupport via a linkage between the polymerase and the solid support orvia a linkage between the next correct nucleotide and the solid support;and (b) separating the solid support from the mixture of nucleic acids,thereby separating the target alleles from the mixture of nucleic acids.

The disclosure further provides a method for separating first alleles ata plurality of loci from second alleles at the plurality of loci,respectively. The method can include steps of (a) providing a mixture ofthe second alleles at the plurality of loci, respectively, in fluidiccontact with a plurality of stabilized ternary complexes that are solidsupport-attached, wherein the stabilized ternary complexes each includea polymerase, primed nucleic acid template, and next correct nucleotide,wherein the template includes a first allele, wherein the next correctnucleotide is a cognate nucleotide for the first allele or the 3′ end ofthe primer includes a cognate nucleotide for the first allele, andwherein each of the stabilized ternary complexes is attached to thesolid support via a linkage between the polymerase and the solid supportor via a linkage between the next correct nucleotide and the solidsupport; and (b) separating the solid support from the mixture ofnucleic acids, thereby separating the first alleles from the secondalleles at the plurality of loci.

A particularly useful solid support for use in multiplex embodiments isone having an array of features. For example, each stabilized ternarycomplex can be attached to an array via a linkage to a particularfeature of the array, thereby separating templates comprising differentalleles from each other. Any of a variety of arrays known in the art canbe modified for use in a method or composition set forth herein. Forexample, linkages made from commercial arrays (or other nucleic acidarrays) to nucleic acid probes can be replaced with linkages set forthherein for attaching polymerases or nucleotides to surfaces. In otherembodiments, polymerases or nucleotides can be attached tooligonucleotide moieties that are complementary to probes located onnucleic acid arrays. In such embodiments, the nucleotide or polymerasecan be attached to the surface via hybridization or crosslinking of thecomplementary strands. If desired a template and/or primer can beattached to a feature of an array.

Exemplary array substrates that can be useful include, withoutlimitation, a BeadChip™ Array available from Illumina, Inc. (San Diego,Calif.) or arrays such as those described in U.S. Pat. Nos. 6,266,459;6,355,431; 6,770,441; 6,859,570; or 7,622,294; or PCT Publication No. WO00/63437, each of which is incorporated herein by reference. Furtherexamples of commercially available array substrates that can be usedinclude, for example, an Affymetrix GeneChip™ array. A spotted arraysubstrate can also be used according to some embodiments. An exemplaryspotted array is a CodeLink™ Array available from Amersham Biosciences.Another array that is useful is one that is manufactured using inkjetprinting methods such as SurePrint™ Technology available from AgilentTechnologies.

Other useful array substrates include those that are used in nucleicacid sequencing applications. For example, arrays that are used toattach amplicons of genomic fragments (often referred to as clusters)can be particularly useful. Examples of substrates that can be modifiedfor use herein include those described in Bentley et al., Nature456:53-59 (2008), PCT Pub. Nos. WO 91/06678; WO 04/018497 or WO07/123744; U.S. Pat. Nos. 7,057,026; 7,211,414; 7,315,019; 7,329,492 or7,405,281; or U.S. Pat. App. Pub. No. 2008/0108082, each of which isincorporated herein by reference.

A multiplex embodiment can be carried out using allele-specificnucleotides and locus primers of the type exemplified in FIG. 1A. Morespecifically, the next correct nucleotide that is present in astabilized ternary complex can be a cognate nucleotide for a firstallele at a locus such that the next correct nucleotide is not a cognatenucleotide for a second allele at the locus. Loci having alleles thatspan a variety of different nucleotide types can be treatedsequentially. For example, the plurality of loci present in a multiplexformat can span four different types of nucleotides. In this example, afirst iteration of the multiplex method can be carried out to form aplurality of stabilized ternary complexes having cognate nucleotides foronly a first type of the four different types of nucleotides. Then asecond iteration of the multiplex method can be carried out to form aplurality of stabilized ternary complexes having cognate nucleotides foronly a second type of the four different types of nucleotides, thesecond type being different from the first type. In a third iteration, aplurality of stabilized ternary complexes can be formed having cognatenucleotides for only a third type of the four different types ofnucleotides, the third type being different from the first and secondtypes. In a fourth iteration, a plurality of stabilized ternarycomplexes can be formed having cognate nucleotides for only a fourthtype of the four different types of nucleotides, the fourth type beingdifferent from the first, second and third types.

In some multiplex embodiments, allele-specific primers of the typeexemplified in FIG. 1A can be used. More specifically, the 3′ end ofeach primer can have a cognate nucleotide for a first allele at a locussuch that the 3′ end of the primer does not have a cognate nucleotidefor a second allele at the locus. An advantage of using anallele-specific primer in a multiplex format is that loci having allelesthat span a variety of different nucleotide types can be treated inparallel. Because alleles are distinguished by the presence or absenceof cognate nucleotides at the 3′ ends of the primers and not by the nextcorrect nucleotide, a mixture of ternary complexes with different lociand alleles can be in simultaneous contact with more than one type ofnext correct nucleotides. Of course, if desired, a mixture can include aplurality ternary complexes that have been formed with allele-specificprimers and only a subset of the next correct nucleotide types thatcould have formed stabilized ternary complexes in the mixture. Thesubset can include cognate nucleotides for no more than 1, 2, 3 or 4different types of nucleotides in the plurality of loci that arepresent.

One or more template nucleic acids that are captured using a method ofthe present disclosure can be used in a variety of subsequentapplications. For example, the template nucleic acid(s) can be used in apreparative method such as cloning of a gene or gene fragment. Thetemplate can be amplified using a method such as polymerase chainreaction (PCR), rolling circle amplification (RCA), multipledisplacement amplification (MDA) or the like. In some cases, the primerthat was used to form the ternary complex in a capture method can alsobe used for amplification. Generally, a template that is captured usinga method set forth herein can be manipulated using methods known in theart including, but not limited to, those described in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring HarborLaboratory, New York (2001) or in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1998), each ofwhich is incorporated herein by reference.

In particular embodiments, one or more template nucleic acid(s) that arecaptured using a method set forth herein can be used in an analyticalmethod, for example, real time polymerase chain reaction (rtPCR),quantitative PCR (qPCR), nucleic acid sequencing, genotyping analysis,expression analysis or the like. Several of these methods employ a stepof extending a primer along a template to be analyzed. In some cases,the primer that was used to form the ternary complex in a capture methodcan also be used for primer extension in an analytical technique.

Optionally, sequencing is carried out as described in commonly ownedU.S. Ser. No. 14/805,381, now published as U.S. Pub. No. 2017/0022553A1, which is incorporated herein by reference. Briefly, methods fordetermining the sequence of a template nucleic acid molecule can bebased on formation of a ternary complex (between polymerase, primednucleic acid and cognate nucleotide) under specified conditions. Themethod can generally include an examination step prior to incorporationof a nucleotide. The examination step can involve providing a templatenucleic acid molecule primed with a primer; contacting the primedtemplate nucleic acid molecule with a first reaction mixture thatincludes a polymerase and at least one nucleotide molecule; monitoringthe interaction of the polymerase with the primed template nucleic acidmolecule in the presence of the nucleotide molecule, without chemicalincorporation of the nucleotide molecule into the primed templatenucleic acid; and identifying a next base in the template nucleic acidusing the monitored interaction of the polymerase with the primedtemplate nucleic acid molecule in the presence of the nucleotidemolecule. In this procedure, ternary complex stabilization and binarycomplex destabilization advantageously enhance discrimination betweencorrect and incorrect nucleotides.

Sequencing-by-synthesis (SBS) techniques can also be used. SBS generallyinvolves the enzymatic extension of a nascent primer through theiterative addition of nucleotides against a template strand to which theprimer is hybridized. SBS can utilize nucleotide monomers that have aterminator moiety or those that lack any terminator moieties. Methodsutilizing monomers having terminators include, for example, thosedescribed in WO 04/018497, U.S. Pat. No. 7,057,026, WO 91/106678, WO07/123744, U.S. US 2007/0166705, US 2006/0188901, US 2006/0240439, US2006/0281109, WO 05/065814, US 2005/0100900, WO 06/064199 or WO07010251, the disclosures of which are incorporated herein by reference.Also useful are SBS methods that are commercially available fromIllumina, Inc., San Diego, Calif.

Some SBS embodiments include detection of a proton released uponincorporation of a nucleotide into an extension product. For example,sequencing based on detection of released protons can use an electricaldetector and associated techniques that are commercially available fromThermo Fisher (Waltham, Mass.) or sequencing methods and systemsdescribed in US Pat. App. Pub. Nos. 2009/0026082 A1; 2009/0127589 A1;2010/0137143 A1; or 2010/0282617 A1, each of which is incorporatedherein by reference.

Other sequencing procedures can be used, such as pyrosequencing.Pyrosequencing detects the release of inorganic pyrophosphate (PPi) asparticular nucleotides are incorporated into a nascent primer hybridizedto a template nucleic acid strand (Ronaghi, et al., AnalyticalBiochemistry 242 (1), 84-9 (1996); Ronaghi, Genome Res. 11 (1), 3-11(2001); Ronaghi et al. Science 281 (5375), 363 (1998); U.S. Pat. Nos.6,210,891; 6,258,568 and 6,274,320, each of which is incorporated hereinby reference). In pyrosequencing, released PPi can be detected by beingconverted to adenosine triphosphate (ATP) by ATP sulfurylase, and theresulting ATP can be detected via luciferase-produced photons. Thus, thesequencing reaction can be monitored via a luminescence detectionsystem.

Sequencing-by-ligation reactions are also useful including, for example,those described in Shendure et al. Science 309:1728-1732 (2005); U.S.Pat. Nos. 5,599,675; and 5,750,341, each of which is incorporated hereinby reference. Some embodiments can include sequencing-by-hybridizationprocedures as described, for example, in Bains et al., Journal ofTheoretical Biology 135 (3), 303-7 (1988); Drmanac et al., NatureBiotechnology 16, 54-58 (1998); Fodor et al., Science 251 (4995),767-773 (1995); and WO 1989/10977, each of which is incorporated hereinby reference. In both sequencing-by-ligation andsequencing-by-hybridization procedures, primers that are hybridized tonucleic acid templates are subjected to repeated cycles of extension byoligonucleotide ligation. Typically, the oligonucleotides arefluorescently labeled and can be detected to determine the sequence ofthe template.

Some embodiments can utilize methods involving the real-time monitoringof DNA polymerase activity. For example, nucleotide incorporations canbe detected through fluorescence resonance energy transfer (FRET)interactions between a fluorophore-bearing polymerase andgamma-phosphate-labeled nucleotides, or with zeromode waveguides (ZMW).Techniques and reagents for sequencing via FRET and or ZMW detection aredescribed, for example, in Levene et al. Science 299, 682-686 (2003);Lundquist et al. Opt. Lett. 33, 1026-1028 (2008); Korlach et al. Proc.Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which areincorporated herein by reference.

In some embodiments, sequencing methods utilize a polymerase that isattached to a ZMW or other solid-support feature. A ternary complex thatis captured in a method set forth herein, or a component thereof, can beattached to a ZMW or other solid support used in sequencing techniquesset forth above or otherwise known in the art.

Another useful application for a template nucleic acid captured by amethod of the present disclosure is gene expression analysis. Geneexpression can be detected or quantified using RNA sequencingtechniques, such as those, referred to as digital RNA sequencing. RNAsequencing techniques can be carried out using sequencing methodologiesknown in the art such as those set forth above. Gene expression can alsobe detected or quantified using hybridization techniques carried out bydirect hybridization to an array or using a multiplex assay, theproducts of which are detected on an array. An array of the presentdisclosure can also be used to determine genotypes for a genomic DNAsample from one or more individual. Exemplary methods for array-basedexpression and genotyping analysis that can be carried out using atemplate nucleic acid captured by methods of the present disclosure aredescribed in U.S. Pat. Nos. 7,582,420; 6,890,741; 6,913,884 or 6,355,431or US Pub. Nos. 2005/0053980; 2009/0186349 or 2005/0181440, each ofwhich is incorporated herein by reference.

Furthermore, nucleic acids that are separated using a method set forthherein can be detected in a method set forth in U.S. ProvisionalApplication No. 62/448,630, filed Jan. 20, 2017, US Pat. App. Docket No.OMNI-014 (097128-1036629 (022PV1), having the title “GENOTYPING BYPOLYMERASE BINDING,” filed concurrently with the present application,and incorporated herein by reference in its entirety. More specifically,one or more alleles that are separated using a method set forth hereincan be detected in a polymerase-based method for detecting oridentifying target alleles of interest. Embodiments of the methodsexploit the specificity with which a polymerase can form a stabilizedternary complex with a primed template and a next correct nucleotide.For example, a stabilized ternary complex can be formed between apolymerase, primed template having a target allele and cognatenucleotide for the allele. An advantage of the methods is thatpolymerase specificity allows a target allele to be distinguished fromother nucleic acids, including for example, other alleles that differfrom the target allele, in some cases by only a single nucleotide. Forexample, a ternary complex can be formed between a polymerase, a primedtemplate encoding a target single nucleotide polymorphism (SNP) alleleand a cognate nucleotide for the SNP allele. Detection of the ternarycomplex will result in selective detection of the SNP allele, comparedto a non-target SNP allele at the same locus, because the cognatenucleotide is selective for the target SNP when forming a ternarycomplex with the polymerase.

Methods and compositions set forth herein can be used to detect rarealleles (e.g. DNA- or RNA-based) containing various mutations withintheir sequences. The methods are well suited to detect even rare variantalleles from pools of purified or semi-purified oligonucleotidescontaining wild-type DNA sequences of the same locus, as well as otherunrelated sequences. Useful primer-nucleotide combinations that can beused to form allele-specific ternary complexes in a detection methodinclude those

shown in FIG. 1A and FIG. 1B. In an exemplary embodiment, a firstallele-specific primer can be present at a first feature of an array anda second allele-specific primer can be present at a second feature ofthe array. The array can be contacted with template nucleic acids,polymerases and a mixture of four different nucleotide types all havingthe same label. A mismatch between the primer and nucleic acid templatewill inhibit polymerase binding, whereas a matched primer template canbind polymerase and a next correct nucleotide to form a stabilizedternary complex. Optionally the array can be washed. The array can thenbe detected using a device that spatially resolves the features andsenses the presence or absence of the labels.

An alternative array format for detection of alleles can utilizelocus-specific primers and allele-specific cognate nucleotides that aredistinguishably labelled. In the first step, nucleic acid templateshaving different alleles can be hybridized to a feature on an arrayhaving multiple copies of the locus-specific primer. As such, twodifferent alleles can bind at the feature. Polymerases can then be boundto the primer-template hybrids in the presence of two differentnucleotide types having distinct labels to form stabilized ternarycomplexes. Optionally the array can be washed. The array can then bedetected using a device that distinguishes the two labels.

Accordingly, one or more allele(s) separated in a method set forthherein can be detected in a method that follows.

A method for identifying target alleles can include steps of (a) forminga plurality of stabilized ternary complexes at a plurality of featureson an array, wherein the stabilized ternary complexes each has apolymerase, a template nucleic acid having a target allele of a locus, aprimer hybridized to the locus, and a next correct nucleotide having acognate in the locus, wherein either (i) the primer is anallele-specific primer having a 3′ nucleotide that is a cognatenucleotide for the target allele, or (ii) the primer is a locus-specificprimer and the next correct nucleotide hybridizes to the target allele;and (b) detecting stabilized ternary complexes at the features, therebyidentifying the target alleles.

In some embodiments, the method for identifying target alleles caninclude steps of (a) providing an array of features, wherein differentlocus-specific primers are attached at different features of the array;(b) contacting the array with a plurality of nucleic acid templates,polymerases and nucleotides to form a plurality of stabilized ternarycomplexes at a plurality of the features, wherein the stabilized ternarycomplexes each has a polymerase, template nucleic acid having a targetallele of a locus, a locus-specific primer hybridized to the locus, anda next correct nucleotide that is a cognate to the target allele; and(c) detecting stabilized ternary complexes at the features, therebyidentifying the target alleles.

An alternative embodiment of the method for identifying target allelescan include steps of (a) providing an array of features, whereindifferent allele-specific primers are attached at different features ofthe array; (b) contacting the array with a plurality of nucleic acidtemplates, polymerases and nucleotides to form a plurality of stabilizedternary complexes at a plurality of the features, wherein the stabilizedternary complexes each has a polymerase, template nucleic acid having atarget allele of a locus, an allele-specific primer hybridized to thelocus, and a next correct nucleotide having a cognate in the locus,wherein the 3′ end of the allele-specific primer has a cognatenucleotide for the target allele; and (c) detecting stabilized ternarycomplexes at the features, thereby identifying the target alleles.

A method for identifying target alleles in a mixture of nucleic acidscan include steps of (a) providing an array of features, whereindifferent locus-specific primers are attached at a first subset of thefeatures of the array, and wherein different allele-specific primers areattached at a second subset of the features of the array; (b) contactingthe array with a plurality of nucleic acid templates, polymerases andnucleotides to form a plurality of stabilized ternary complexes at aplurality of the features, wherein the stabilized ternary complexes atthe first subset of features each has a polymerase, template nucleicacid having a target allele of a locus, a locus-specific primerhybridized to the locus, and a next correct nucleotide that is a cognateto the target allele, wherein the stabilized ternary complexes at thesecond subset of features each has a polymerase, template nucleic acidhaving a target allele of a locus, an allele-specific primer hybridizedto the locus, and a next correct nucleotide having a cognate in thelocus, and wherein the 3′ end of the allele-specific primer has acognate nucleotide for the target allele; and (c) detecting stabilizedternary complexes at the features, thereby identifying the targetalleles.

Also provided is a method for identifying target alleles in a mixture ofnucleic acids, that includes steps of (a) providing an array offeatures, wherein different template nucleic acids are attached atdifferent features of the array; (b) contacting the array with aplurality of primers, polymerases and nucleotides to form a plurality ofstabilized ternary complexes at a plurality of the features, wherein thestabilized ternary complexes at the features each has a polymerase, atemplate nucleic acid attached to a feature of the array and having atarget allele of a locus, a primer hybridized to the locus, and a nextcorrect nucleotide having a cognate in the locus, wherein either (i) theprimer is an allele-specific primer having a 3′ nucleotide that is acognate nucleotide for the target allele, or (ii) the primer is alocus-specific primer and the next correct nucleotide hybridizes to thetarget allele; and (c) detecting stabilized ternary complexes at thefeatures, thereby identifying the target alleles.

Further provided is a method for identifying target alleles in a mixtureof nucleic acids that includes steps of (a) providing an array offeatures, wherein polymerases are attached at features of the array; (b)contacting the array with a plurality of primers, template nucleic acidsand nucleotides to form a plurality of stabilized ternary complexes at aplurality of the features, wherein the stabilized ternary complexes atthe features each has a polymerase that is attached at a feature of thearray, template nucleic acid having a target allele of a locus, a primerhybridized to the locus, and a next correct nucleotide having a cognatein the locus, wherein either (i) the primer is an allele-specific primerhaving a 3′ nucleotide that is a cognate nucleotide for the targetallele, or (ii) the primer is a locus-specific primer and the nextcorrect nucleotide hybridizes to the target allele; and (c) detectingstabilized ternary complexes at the features, thereby identifying thetarget alleles.

EXAMPLE 1 Distinguishing the Next Correct Nucleotide From a MismatchedNucleotide.

Methods & Materials. Polymerase buffer: 20 mM Tris, pH 8, 300 mM NaCl, 5mM DTT, 100 μM dNTP, 150 nM Kienow, 0.01% BSA, 0.02% Tween-20, 10 mMMgCl₂. Exam buffer: 20 mM Tris, pH 8, 300 mM NaCl, 5 mM DTT, 100 μMdNTP, 150 nM Klenow, 0.01% BSA, 0.02% Tween-20. Incorporation buffer: 20mM Tris, pH 8, 300 mM NaCl, 5 mM DTT, 0.01% BSA, 0.02% Tween-20, 10 mMMgCl₂. Wash Buffer: 20 mM Tris, pH 8, 300 mM NaCl, 5 mM DTT, 0.01% BSA,0.02% Tween-20.

FIG. 4 shows the results of a binding assay using polymerase, primedtemplate, and nucleotide (either matched or mismatched with the nextbase in the template), where magnesium was present or absent during thebinding assay. The first delivered nucleotide was dCTP (C:T mismatch)and the second delivery was dATP (A:T match). The solid line in FIG. 3shows the results with Polymerase buffer. The pre-steady state rateconstants were 0.0106 and 0.0084 for the match A and mismatch C steps,respectively. The difference was too small to accurately discriminatethe cognate base. The dashed line in FIG. 4 represents a magnesium freebinding step in Exam buffer, followed by soaking in incorporationbuffer. A signal threshold of 1.1 nm allowed accurate identification ofthe correct base. These results show that the sensing platform wasunable to discriminate a match from mismatch base when magnesium wasincluded in the buffer during examination (Polymerase Buffer, solidline, FIG. 4). In contrast, binding in the absence of magnesium providedvery large discrimination between correct and incorrect base (ExamBuffer, dashed line, FIG. 4). The correct base sequence was determinedby signal thresholding rather than binding rates.

EXAMPLE 2 Effect of Salt Concentration on Match/Mismatch BaseDiscrimination

The FORTEBIO® Octet instrument (Red384 or qk) (Menlo Park, Calif.) usesbiolayer interferometry to measure binding reactions at the surface of afiber optic tip. In this example, the tips were functionalized withstreptavidin (SA) to enable binding to 5′ biotin labeled DNA templateshybridized with a primer that is complementary to sequences near the 3′end of the template.

Experimental Conditions: PhiX_matchC and phiX_matchA were loaded ontoindividual tips. Primer-template was loaded onto the tips at between 100and 500 nM in 1-2× PBS containing 0.01-0.02% BSA and 0.01-0.02% Tween 20(loading buffer). The FP2 primer was in 1.25-2 fold excess overtemplate. Loading was monitored by change in signal and usually reacheda plateau within 5 minutes at 30 degrees C. Tips were soaked in Loadingbuffer for 1-5 minutes to remove unbound DNA material. For base calling,the tips were soaked in solutions containing IX Taq buffer (10 mMTris-HCl, 50 mM KCl, pH 8.3, 25° C., magnesium free) supplemented with0.01-0.02% BSA and 0.01-0.02% Tween 20 (LS buffer), 100 nM polymeraseenzyme, 100 μM NTP, and varying concentrations of additional NaCl from50 to 300 mM. The phiX_matchC duplex will form a ternary complex andshow an increase in binding signal because the next correct nucleotide(cognate) is presented. The phiX_matchA should not because it is anincorrect nucleotide (noncognate).

Results: At standard reaction conditions both templates bound polymeraseenzyme. However, as the salt concentration increased the bindingaffinity of the noncognate complex decreased while binding affinity ofthe cognate complex remained high. Thus, the signal to noise ratio (SNR)of base discrimination increased such that the next correct base waseasily identified during this examination step (FIG. 5). Sodium chloride(NaCl) was used in this example but salts such as KCl, NH₂(SO₄),potassium glutamate, and others known in the art can be used.Polymerases that show differences in binding affinity between correctand incorrect nucleotides included Klenow, Bst2.0, Bsu, and Taq.

Throughout this application various publications, patents and/or patentapplications have been referenced. The disclosures of these documents intheir entireties are hereby incorporated by reference in thisapplication.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made. Accordingly, otherembodiments are within the scope of the following claims.

What is claimed is:
 1. A method for separating a plurality of targetalleles from a mixture of nucleic acids, comprising (a) providing amixture of nucleic acids in fluidic contact with an array of stabilizedternary complexes, wherein each of the stabilized ternary complexes isattached to a feature of the array, wherein the stabilized ternarycomplexes each comprises a polymerase, primed nucleic acid template, andnext correct nucleotide, wherein the template comprises a target allele,wherein the next correct nucleotide is a cognate nucleotide for thetarget allele, and wherein each of the stabilized ternary complexes isattached to the array via a linkage between the polymerase and a featureof the array or via a linkage between the next correct nucleotide and afeature of the array; and (b) separating the array from the mixture ofnucleic acids, thereby separating the target alleles from the mixture ofnucleic acids.
 2. The method of claim 1, wherein each of the stabilizedternary complexes is attached to the array via a linkage between thepolymerase and a feature of the array.
 3. The method of claim 1, whereinone or more of the target alleles occurs at a locus and the mixture ofnucleic acids further comprises another allele at the locus.
 4. Themethod of claim 3, wherein the separating of the array from the mixtureof nucleic acids thereby separates the target alleles from the otheralleles.
 5. The method of claim 3, wherein at least one of the targetalleles is a minor allele.
 6. The method of claim 5, wherein the minorallele frequency is less than 5%.
 7. The method of claim 1, furthercomprising (c) dissociating the templates comprising the target allelesfrom the separated array.
 8. The method of claim 7, further comprising(d) forming a mixture comprising second stabilized ternary complexesthat comprise the formerly dissociated templates, polymerases, and nextcorrect nucleotides that are cognate nucleotides for the target alleles,wherein each of the second stabilized ternary complexes is attached toan array via a linkage between the polymerase and a feature of the arrayor via a linkage between the next correct nucleotide and a feature ofthe array.
 9. The method of claim 8, further comprising (e) separatingthe array of step (d) from the mixture, thereby separating the formerlydissociated templates comprising the target alleles from the mixture.10. The method of claim 9, further comprising (f) dissociating theformerly dissociated templates from the array separated in step (e). 11.The method of claim 8, wherein the solid support of step (a) is the sameas the solid support of step (d).
 12. The method of claim 10, whereinthe polymerase or the next correct nucleotide of step (a) is the same asthe polymerase or the next correct nucleotide of step (d).
 13. Themethod of claim 1, wherein step (a) further comprises providing amixture of nucleic acids comprising the templates and contacting themixture with the polymerases and the next correct nucleotides, therebyproviding the mixture of nucleic acids in fluidic contact with the arrayof stabilized ternary complexes.
 14. The method of claim 13, wherein thepolymerases or next correct nucleotides are attached to a feature of thearray prior to forming the stabilized ternary complexes.
 15. The methodof claim 13, wherein the stabilized ternary complexes are formed insolution prior to attaching the ternary complexes to the array.
 16. Themethod of claim 1, wherein the linkage to the array comprises areceptor-ligand association.
 17. The method of claim 16, wherein step(a) further comprises forming the stabilized ternary complexes insolution and then binding the receptor to the ligand, thereby attachingthe ternary complexes to the array.
 18. The method of claim 16, whereinstep (a) further comprises providing the polymerase attached to thearray and then forming the stabilized ternary complexes on the array.19. The method of claim 16, wherein step (a) further comprises providingthe next correct nucleotides attached to the array and then forming thestabilized ternary complexes on the array.
 20. The method of claim 1,wherein the ternary complexes lack exogenous labels.